Cycas Taitungensis Research Articles

Structural and gene composition variation of the complete mitochondrial genome of Mammillaria huitzilopochtli (Cactaceae, Caryophyllales), revealed by de novo assembly

BackgroundStructural descriptions of complete genomes have elucidated evolutionary processes in angiosperms. In Cactaceae (Caryophyllales), a high structural diversity of the chloroplast genome has been identified within and among genera. In this study, we assembled the first mitochondrial genome (mtDNA) for the short-globose cactus Mammillaria huitzilopochtli. For comparative purposes, we used the published genomes of 19 different angiosperms and the gymnosperm Cycas taitungensis as an external group for phylogenetic issues.ResultsThe mtDNA of M. huitzilopochtli was assembled into one linear chromosome of 2,052,004 bp, in which 65 genes were annotated. These genes account for 57,606 bp including 34 protein-coding genes (PCGs), 27 tRNAs, and three rRNAs. In the non-coding sequences, repeats were abundant, with a total of 4,550 (179,215 bp). In addition, five complete genes (psaC and four tRNAs) of chloroplast origin were documented. Negative selection was estimated for most (23) of the PCGs. The phylogenetic tree showed a topology consistent with previous analyses based on the chloroplast genome.ConclusionsThe number and type of genes contained in the mtDNA of M. huitzilopochtli were similar to those reported in 19 other angiosperm species, regardless of their phylogenetic relationships. Although other Caryophyllids exhibit strong differences in structural arrangement and total size of mtDNA, these differences do not result in an increase in the typical number and types of genes found in M. huitzilopochtli. We concluded that the total size of mtDNA in angiosperms increases by the lengthening of the non-coding sequences rather than a significant gain of coding genes.

The complete mitochondrial genome of Cycas debaoensis revealed unexpected static evolution in gymnosperm species.

Mitochondrial genomes of vascular plants are well known for their liability in architecture evolution. However, the evolutionary features of mitogenomes at intra-generic level are seldom studied in vascular plants, especially among gymnosperms. Here we present the complete mitogenome of Cycas debaoensis, an endemic cycad species to the Guangxi region in southern China. In addition to assemblage of draft mitochondrial genome, we test the conservation of gene content and mitogenomic stability by comparing it to the previously published mitogenome of Cycas taitungensis. Furthermore, we explored the factors such as structural rearrangements and nuclear surveillance of double-strand break repair (DSBR) proteins in Cycas in comparison to other vascular plant groups. The C. debaoensis mitogenome is 413,715 bp in size and encodes 69 unique genes, including 40 protein coding genes, 26 tRNAs, and 3 rRNA genes, similar to that of C. taitungensis. Cycas mitogenomes maintained the ancestral intron content of seed plants (26 introns), which is reduced in other lineages of gymnosperms, such as Ginkgo biloba, Taxus cuspidata and Welwitschia mirabilis due to selective pressure or retroprocessing events. C. debaoensis mitogenome holds 1,569 repeated sequences (> 50 bp), which partially account for fairly large intron size (1200 bp in average) of Cycas mitogenome. The comparison of RNA-editing sites revealed 267 shared non-silent editing site among predicted vs. empirically observed editing events. Another 33 silent editing sites from empirical data increase the total number of editing sites in Cycas debaoensis mitochondrial protein coding genes to 300. Our study revealed unexpected conserved evolution between the two Cycas species. Furthermore, we found strict collinearity of the gene order along with the identical set of genomic content in Cycas mt genomes. The stability of Cycas mt genomes is surprising despite the existence of large number of repeats. This structural stability may be related to the relative expansion of three DSBR protein families (i.e., RecA, OSB, and RecG) in Cycas nuclear genome, which inhibit the homologous recombinations, by monitoring the accuracy of mitochondrial chromosome repair.

Genetic evidence of the southward founder speciation of Cycas taitungensis from ancestral C. revoluta along the Ryukyu Archipelagos

Cycas taitungensis and its sister species, C. revoluta (Cycas sect. Asiorientalis), are distributed peripatrically. Previous studies have revealed their phylogeographic pattern and, based on certain plastid DNA markers, have established that they diverged recently. However, the speciation process involving the genomic divergence has not been well clarified. In this study, based on their geographical distribution and estimates of their genetic diversity, two speciation models are proposed: (1) the founder and (2) the bottleneck speciation mode. Using approximate Bayesian computation (ABC) in combination with their estimated genetic diversity and population structure, we infer that the ancestral C. taitungensis populations diverged and southward colonized southeastern Taiwan from ancestral C. revoluta since the middle Pleistocene. These founders preserved ancestral polymorphism, resulting in small differences in genetic diversity and effective population size (Ne), despite a large difference in census population size (Nc) between two species. Based on this case of island cycad species offshore of continental Asia, this study provides new insight into how the speciation process influences the genetic diversity pattern of species with small Nc and Ne.

Diversity in Cycas (Cycadales: Cycadaceae) Species Offered as Larval Food Influences Fecundity of Chilades pandava (Lepidoptera: Lycaenidae) Adults.

Chilades pandava (Lepidoptera: Lycaenidae) larval food quality was studied to determine its influence on adult life history traits. A wild population from Cycas nongnoochiae (Cycadales: Cycadaceae) endemic habitat behaved similarly to the population collected from a garden setting. Cycas micronesica, Cycas revoluta, and Cycas seemannii leaves were used as high-quality food, whereas C nongnoochiae, Cycas taitungensis, and Cycas condaoensis leaves were used as low-quality food. The daily oviposition rate was not influenced by food quality, but longevity and lifetime fecundity of females were increased by high-quality larval food. These results indicate that in situ Cycas species impose a physiological constraint on the genetic capacity to produce offspring by C pandava. The removal of that constraint by high-quality novel Cycas species may be one reason this butterfly can increase in population rapidly after an invasion event and express greater herbivory of Cycas species within invaded regions.

Ginkgo and Welwitschia Mitogenomes Reveal Extreme Contrasts in Gymnosperm Mitochondrial Evolution.

Mitochondrial genomes (mitogenomes) of flowering plants are well known for their extreme diversity in size, structure, gene content, and rates of sequence evolution and recombination. In contrast, little is known about mitogenomic diversity and evolution within gymnosperms. Only a single complete genome sequence is available, from the cycad Cycas taitungensis, while limited information is available for the one draft sequence, from Norway spruce (Picea abies). To examine mitogenomic evolution in gymnosperms, we generated complete genome sequences for the ginkgo tree (Ginkgo biloba) and a gnetophyte (Welwitschia mirabilis). There is great disparity in size, sequence conservation, levels of shared DNA, and functional content among gymnosperm mitogenomes. The Cycas and Ginkgo mitogenomes are relatively small, have low substitution rates, and possess numerous genes, introns, and edit sites; we infer that these properties were present in the ancestral seed plant. By contrast, the Welwitschia mitogenome has an expanded size coupled with accelerated substitution rates and extensive loss of these functional features. The Picea genome has expanded further, to more than 4 Mb. With regard to structural evolution, the Cycas and Ginkgo mitogenomes share a remarkable amount of intergenic DNA, which may be related to the limited recombinational activity detected at repeats in Ginkgo Conversely, the Welwitschia mitogenome shares almost no intergenic DNA with any other seed plant. By conducting the first measurements of rates of DNA turnover in seed plant mitogenomes, we discovered that turnover rates vary by orders of magnitude among species.

Evolutionary Stasis in Cycad Plastomes and the First Case of Plastome GC-Biased Gene Conversion.

In angiosperms, gene conversion has been known to reduce the mutational load of plastid genomes (the plastomes). Particularly, more frequent gene conversions in inverted repeat (IR) than in single copy (SC) regions result in contrasting substitution rates between these two regions. However, little has been known about the effect of gene conversion in the evolution of gymnosperm plastomes. Cycads (Cycadophyta) are the second largest gymnosperm group. Evolutionary study of their plastomes is limited to the basal cycad genus, Cycas. In this study, we addressed three questions. 1) Do the plastomes of other cycad genera evolve slowly as previously observed in the plastome of Cycas taitungensis? 2) Do substitution rates differ between their SC and IR regions? And 3) Does gene conversion occur in the cycad plastomes? If yes, is it AT-biased or GC-biased? Plastomes of eight species from other eight genera of cycads were sequenced. These plastomes are highly conserved in genome organization. Excluding ginkgo, cycad plastomes have significantly lower synonymous and nonsynonymous substitution rates than other gymnosperms, reflecting their evolutionary stasis in nucleotide mutations. In the IRs of cycad plastomes, the reduced substitution rates and GC-biased mutations are associated with a GC-biased gene conversion (gBGC) mechanism. Further investigations suggest that in cycads, gBGC is able to rectify plastome-wide mutations. Therefore, this study is the first to uncover the plastomic gBGC in seed plants. We also propose a gBGC model to interpret the dissimilar evolutionary patterns as well as the compositionally biased mutations in the SC and IR regions of cycad plastomes.

The complete mitochondrial genome of Gossypium hirsutum and evolutionary analysis of higher plant mitochondrial genomes.

BackgroundMitochondria are the main manufacturers of cellular ATP in eukaryotes. The plant mitochondrial genome contains large number of foreign DNA and repeated sequences undergone frequently intramolecular recombination. Upland Cotton (Gossypium hirsutum L.) is one of the main natural fiber crops and also an important oil-producing plant in the world. Sequencing of the cotton mitochondrial (mt) genome could be helpful for the evolution research of plant mt genomes.Methodology/Principal FindingsWe utilized 454 technology for sequencing and combined with Fosmid library of the Gossypium hirsutum mt genome screening and positive clones sequencing and conducted a series of evolutionary analysis on Cycas taitungensis and 24 angiosperms mt genomes. After data assembling and contigs joining, the complete mitochondrial genome sequence of G. hirsutum was obtained. The completed G.hirsutum mt genome is 621,884 bp in length, and contained 68 genes, including 35 protein genes, four rRNA genes and 29 tRNA genes. Five gene clusters are found conserved in all plant mt genomes; one and four clusters are specifically conserved in monocots and dicots, respectively. Homologous sequences are distributed along the plant mt genomes and species closely related share the most homologous sequences. For species that have both mt and chloroplast genome sequences available, we checked the location of cp-like migration and found several fragments closely linked with mitochondrial genes.ConclusionThe G. hirsutum mt genome possesses most of the common characters of higher plant mt genomes. The existence of syntenic gene clusters, as well as the conservation of some intergenic sequences and genic content among the plant mt genomes suggest that evolution of mt genomes is consistent with plant taxonomy but independent among different species.

The Mitochondrial Genome of Raphanus sativus and Gene Evolution of Cruciferous Mitochondrial Types

To explore the mitochondrial genes of the Cruciferae family, the mitochondrial genome of Raphanus sativus (sat) was sequenced and annotated. The circular mitochondrial genome of sat is 239,723bp and includes 33 protein-coding genes, three rRNA genes and 17 tRNA genes. The mitochondrial genome also contains a pair of large repeat sequences 5.9kb in length, which may mediate genome reorganization into two sub-genomic circles, with predicted sizes of 124.8kb and 115.0kb, respectively. Furthermore, gene evolution of mitochondrial genomes within the Cruciferae family was analyzed using sat mitochondrial type (mitotype), together with six other reported mitotypes. The cruciferous mitochondrial genomes have maintained almost the same set of functional genes. Compared with Cycas taitungensis (a representative gymnosperm), the mitochondrial genomes of the Cruciferae have lost nine protein-coding genes and seven mitochondrial-like tRNA genes, but acquired six chloroplast-like tRNAs. Among the Cruciferae, to maintain the same set of genes that are necessary for mitochondrial function, the exons of the genes have changed at the lowest rates, as indicated by the numbers of single nucleotide polymorphisms. The open reading frames (ORFs) of unknown function in the cruciferous genomes are not conserved. Evolutionary events, such as mutations, genome reorganizations and sequence insertions or deletions (indels), have resulted in the non-conserved ORFs in the cruciferous mitochondrial genomes, which is becoming significantly different among mitotypes. This work represents the first phylogenic explanation of the evolution of genes of known function in the Cruciferae family. It revealed significant variation in ORFs and the causes of such variation.

Temperature-Dependent Demography of <I>Aulacaspis yasumatsui</I> (Hemiptera: Diaspididae)

Aulacaspis yasumatsui Takagi continues to threaten the extinction of the endemic and endangered Cycas taitungensis (Shen et al.) in Taiwan. Failure to understand its population demographic parameters in detail will continue to hinder the success of pest management practices. An in-depth knowledge of the development, survival, and fecundity of A. yasumatsui under different environmental conditions is necessary to understand its' population growth. The demography of A. yasumatsui was studied in the laboratory based on the age-stage, two-sex life table at 20, 23, 25, 28, and 31 degrees C, 70% relative humidity, and a photoperiod of 16:8 (L:D) h. The intrinsic rate of increase (r) under these temperatures was 0.06, 0.07, 0.09, 0.10, and 0.08 d(-1), respectively. The net reproductive rate (R0) was 46.27, 47.78, 69.50, 96.08, and 56.65 offsprings per individual and the mean generation time (T) was 65.60, 55.81, 47.82, 44.15, and 51.42 d, respectively. A. yasumatsui does well at warmer temperatures (25-28 degrees C); however, its performance is disrupted at lower temperatures. This study provides insight on how to minimize growth and destruction of A. yasumatsui and conservation of Cy. taitungensis; new cycad reserves should be set up in cooler areas in Taiwan.

The Complete Chloroplast Genome of Ginkgo biloba Reveals the Mechanism of Inverted Repeat Contraction

We determined the complete chloroplast genome (cpDNA) of Ginkgo biloba (common name: ginkgo), the only relict of ginkgophytes from the Triassic Period. The cpDNA molecule of ginkgo is quadripartite and circular, with a length of 156,945 bp, which is 6,458 bp shorter than that of Cycas taitungensis. In ginkgo cpDNA, rpl23 becomes pseudo, only one copy of ycf2 is retained, and there are at least five editing sites. We propose that the retained ycf2 is a duplicate of the ancestral ycf2, and the ancestral one has been lost from the inverted repeat A (IRA). This loss event should have occurred and led to the contraction of IRs after ginkgos diverged from other gymnosperms. A novel cluster of three transfer RNA (tRNA) genes, trnY-AUA, trnC-ACA, and trnSeC-UCA, was predicted to be located between trnC-GCA and rpoB of the large single-copy region. Our phylogenetic analysis strongly suggests that the three predicted tRNA genes are duplicates of trnC-GCA. Interestingly, in ginkgo cpDNA, the loss of one ycf2 copy does not significantly elevate the synonymous rate (Ks) of the retained copy, which disagrees with the view of Perry and Wolfe (2002) that one of the two-copy genes is subjected to elevated Ks when its counterpart has been lost. We hypothesize that the loss of one ycf2 is likely recent, and therefore, the acquired Ks of the retained copy is low. Our data reveal that ginkgo possesses several unique features that contribute to our understanding of the cpDNA evolution in seed plants.

RNA Editing Sites Exist in Protein-coding Genes in the Chloroplast Genome of Cycas taitungensis

RNA editing is a post-transcriptional process that results in modifications of ribonucleotides at specific locations. In land plants editing can occur in both mitochondria and chloroplasts and most commonly involves C-to-U changes, especially in seed plants. Using prediction and experimental determination, we investigated RNA editing in 40 protein-coding genes from the chloroplast genome of Cycas taitungensis. A total of 85 editing sites were identified in 25 transcripts. Comparison analysis of the published editotypes of these 25 transcripts in eight species showed that RNA editing events gradually disappear during plant evolution. The editing in the first and third codon position disappeared quicker than that in the second codon position. ndh genes have the highest editing frequency while serine and proline codons were more frequently edited than the codons of other amino acids. These results imply that retained RNA editing sites have imbalanced distribution in genes and most of them may function by changing protein structure or interaction. Mitochondrion protein-coding genes have three times the editing sites compared with chloroplast genes of Cycas, most likely due to slower evolution speed.

Maternal inheritance of plastids and mitochondria in Cycas L. (Cycadaceae)

Cycas is often considered a living fossil, thereby providing a unique model for revealing the evolution of spermatophytes. To date, the genetic inheritance of these archaic plants is not fully understood. The present study seeks to document the process of organelle inheritance in an interspecific cross of Cycas species. Extranuclear organelle DNA from chloroplasts and mitochondria was analyzed using both polymerase chain reaction-restriction fragment length polymorphism analysis and microscopy. Here, we show that the chloroplasts and mitochondria in the progeny of interspecific crosses between Cycas taitungensis and Cycas ferruginea were exclusively inherited from the female parent. Epifluorescence microscopic analyses of the pollen cells from Cycas elongata indicated that there was a significant degradation of organelle DNA in male reproductive cells following maturation; the DNA fluorescent signals were only seen after pollen mitosis two, but not detectable at mature stage. Lack of organelle DNA fluorescent signal in prothallial cells was confirmed by the absence of plastids and mitochondria in electronic microscopic images. In conclusion, these data suggest that the maternal plastid and mitochondrial inheritance in Cycas, native to the old world, are the same as seen in seed plants.

Temperature-Dependent Demography of Chilades pandava peripatria (Lepidoptera: Lycaenidae)

Chilades pandava peripatria Hsu and its host plant Cycas taitungensis Shen, Hill, Tsou & Chen are both endemic species to Taiwan. Ch. pandava peripatria has a specific association with buds and soft leaves of cycad plants. The introduced species, Cy. revoluta, have prolonged budding periods and extensive auxiliary buds that extensively contribute to the outbreak of Ch. pandava peripatria. An in-depth knowledge of the development, survival, and fecundity of Ch. pandava peripatria under different environmental conditions is necessary to understand the population growth of Ch. pandava peripatria. The demography of Ch. pandava peripatria was studied based on the age-stage, two-sex life table at 20, 23, 25, 28, and 31 degrees C, 70% RH, and a photoperiod of 16:8 (L:D) h under laboratory conditions. Ch. pandava peripatria completed its development under tested temperatures but did not produce offsprings at 23 degrees C. Because of the high egg mortality at 20 degrees C, the data at this given temperature were excluded from this study. The intrinsic rate of increase (r) under these tested temperatures was 0.1846, 0.2919, and 0.1412 d(-1), respectively. The net reproductive rate (H(o)) was 165.47, 262.32, and 56.68 offsprings per individual and the mean generation time (T) was 27.72, 19.10, and 28.67 d, respectively. Our results indicated that Ch. pandava peripatria is highly adaptable to environments where temperature ranges from 25 to 31 degrees C.

Microsatellite primers in the native perennial cycad Cycas taitungensis (Cycadaceae)

Microsatellite primers were developed for the native perennial cycad Cycas taitungensis to evaluate the genetic variation of this endangered insular species. Using a magnetic bead enrichment method and EST data, 16 primer sets were developed and identified for the native Taiwan cycad C. taitungensis. The primers amplified dinucleotide, trinucleotide, and complex repeats with 1-9 alleles per locus. Most primers also amplified DNA from C. revoluta and C. debaoensis. These results indicate the utility of primers for future studies of the genetic structure of C. taitungensis. In addition, the primers are useful for further phylogeographic studies between C. taitungensis and C. revoluta, which is a closely related species.

Editing site analysis in a gymnosperm mitochondrial genome reveals similarities with angiosperm mitochondrial genomes

Sequence analysis of organelle genomes and comprehensive analysis of C-to-U editing sites from flowering and non-flowering plants have provided extensive sequence information from diverse taxa. This study includes the first comprehensive analysis of RNA editing sites from a gymnosperm mitochondrial genome, and utilizes informatics analyses to determine conserved features in the RNA sequence context around editing sites. We have identified 565 editing sites in 21 full-length and 4 partial cDNAs of the 39 protein-coding genes identified from the mitochondrial genome of Cycas taitungensis. The information profiles and RNA sequence context of C-to-U editing sites in the Cycas genome exhibit similarity in the immediate flanking nucleotides. Relative entropy analyses indicate that similar regions in the 5′ flanking 20 nucleotides have information content compared to angiosperm mitochondrial genomes. These results suggest that evolutionary constraints exist on the nucleotide sequences immediately adjacent to C-to-U editing sites, and similar regions are utilized in editing site recognition.Electronic supplementary materialThe online version of this article (doi:10.1007/s00294-010-0312-4) contains supplementary material, which is available to authorized users.

Life table of cycad scale, Aulacaspis yasumatsui (Hemiptera: Diaspididae), reared on Cycas in Taiwan

Aulacaspis yasumatsui Takagi (Hemiptera: Diaspididae), an invasive scale insect, attacks different species of Cycas (Cycadales: Cycadaceae) in Taiwan. Development, survival and fecundity of A. yasumatsui were studied on Cycas taitungensis at 24 °C, 70 ± 10% RH, and a photoperiod of 12:12 (L:D) h under laboratory conditions. Data on the life history of A. yasumatsui were analyzed using the age-stage, two-sex life table, to address variable development rates among individuals and between sexes. The egg incubation time was 7.26 days for both females and males and female nymphal development duration was 28.65 days. The development duration of male nymphal stages + pupal stage was 19 days. The total pre-oviposition period (TPOP) was 35.92 days. The maximum longevity of female adults was 67 days and 1 day for males. The intrinsic rate of increase ( r) was 0.100 day -1, the finite rate of increase ( λ) was 1.11 day -1, the net reproduction rate ( R o ) was 111.51 offspring /individual, and the mean generation time ( T) was 47.24 day. Life table data can be used to project population growth, to design mass rearing programs and to establish management tactics to control insect pests.

Patterns of synonymous codon usage bias in chloroplast genomes of seed plants

Codon usage in chloroplast genome of six seed plants (Arabidopsis thaliana, Populus alba, Zea mays, Triticum aestivum, Pinus koraiensis and Cycas taitungensis) was analyzed to find general patterns of codon usage in chloroplast genomes of seed plants. The results show that chloroplast genomes of the six seed plants had similar codon usage patterns, with a strong bias towards a high representation of NNA and NNT codons. In chloroplast genomes of the six seed plants, the effective number of codons (ENC) for most genes was similar to that of the expected ENC based on the GC content at the third codon position, but several genes with low ENC values were laying below the expected curve. All of these data indicate that codon usage was dominated by a mutational bias in chloroplast genomes of seed plants and that selection appeared to be limited to a subset of genes and to only subtly affect codon usage. Meantime, four, six, eight, nine, ten and 12 codons were defined as the optimal codons in chloroplast genomes of the six seed plants.

Complete nucleotide sequence of the Cryptomeria japonicaD. Don. chloroplast genome and comparative chloroplast genomics: diversified genomic structure of coniferous species

BackgroundThe recent determination of complete chloroplast (cp) genomic sequences of various plant species has enabled numerous comparative analyses as well as advances in plant and genome evolutionary studies. In angiosperms, the complete cp genome sequences of about 70 species have been determined, whereas those of only three gymnosperm species, Cycas taitungensis, Pinus thunbergii, and Pinus koraiensis have been established. The lack of information regarding the gene content and genomic structure of gymnosperm cp genomes may severely hamper further progress of plant and cp genome evolutionary studies. To address this need, we report here the complete nucleotide sequence of the cp genome of Cryptomeria japonica, the first in the Cupressaceae sensu lato of gymnosperms, and provide a comparative analysis of their gene content and genomic structure that illustrates the unique genomic features of gymnosperms.ResultsThe C. japonica cp genome is 131,810 bp in length, with 112 single copy genes and two duplicated (trnI-CAU, trnQ-UUG) genes that give a total of 116 genes. Compared to other land plant cp genomes, the C. japonica cp has lost one of the relevant large inverted repeats (IRs) found in angiosperms, fern, liverwort, and gymnosperms, such as Cycas and Gingko, and additionally has completely lost its trnR-CCG, partially lost its trnT-GGU, and shows diversification of accD. The genomic structure of the C. japonica cp genome also differs significantly from those of other plant species. For example, we estimate that a minimum of 15 inversions would be required to transform the gene organization of the Pinus thunbergii cp genome into that of C. japonica. In the C. japonica cp genome, direct repeat and inverted repeat sequences are observed at the inversion and translocation endpoints, and these sequences may be associated with the genomic rearrangements.ConclusionThe observed differences in genomic structure between C. japonica and other land plants, including pines, strongly support the theory that the large IRs stabilize the cp genome. Furthermore, the deleted large IR and the numerous genomic rearrangements that have occurred in the C. japonica cp genome provide new insights into both the evolutionary lineage of coniferous species in gymnosperm and the evolution of the cp genome.

The Mitochondrial Genome of the Gymnosperm Cycas taitungensis Contains a Novel Family of Short Interspersed Elements, Bpu Sequences, and Abundant RNA Editing Sites

The mtDNA of Cycas taitungensis is a circular molecule of 414,903 bp, making it 2- to 6-fold larger than the known mtDNAs of charophytes and bryophytes, but similar to the average of 7 elucidated angiosperm mtDNAs. It is characterized by abundant RNA editing sites (1,084), more than twice the number found in the angiosperm mtDNAs. The A + T content of Cycas mtDNA is 53.1%, the lowest among known land plants. About 5% of the Cycas mtDNA is composed of a novel family of mobile elements, which we designated as "Bpu sequences." They share a consensus sequence of 36 bp with 2 terminal direct repeats (AAGG) and a recognition site for the Bpu 10I restriction endonuclease (CCTGAAGC). Comparison of the Cycas mtDNA with other plant mtDNAs revealed many new insights into the biology and evolution of land plant mtDNAs. For example, the noncoding sequences in mtDNAs have drastically expanded as land plants have evolved, with abrupt increases appearing in the bryophytes, and then in the seed plants. As a result, the genomic organizations of seed plant mtDNAs are much less compact than in other plants. Also, the Cycas mtDNA appears to have been exempted from the frequent gene loss observed in angiosperm mtDNAs. Similar to the angiosperms, the 3 Cycas genes nad1, nad2, and nad5 are disrupted by 5 group II intron squences, which have brought the genes into trans-splicing arrangements. The evolutionary origin and invasion/duplication mechanism of the Bpu sequences in Cycas mtDNA are hypothesized and discussed.

Chloroplast Genome (cpDNA) of Cycas taitungensis and 56 cp Protein-Coding Genes of Gnetum parvifolium: Insights into cpDNA Evolution and Phylogeny of Extant Seed Plants

Phylogenetic relationships among the 5 groups of extant seed plants are presently unsettled. To reexamine this long-standing debate, we determine the complete chloroplast genome (cpDNA) of Cycas taitungensis and 56 protein-coding genes encoded in the cpDNA of Gnetum parvifolium. The cpDNA of Cycas is a circular molecule of 163,403 bp with 2 typical large inverted repeats (IRs) of 25,074 bp each. We inferred phylogenetic relationships among major seed plant lineages using concatenated 56 protein-coding genes in 37 land plants. Phylogenies, generated by the use of 3 independent methods, provide concordant and robust support for the monophylies of extant seed plants, gymnosperms, and angiosperms. Within the modern gymnosperms are 2 highly supported sister clades: Cycas-Ginkgo and Gnetum-Pinus. This result agrees with both the "gnetifer" and "gnepines" hypotheses. The sister relationships in Cycas-Ginkgo and Gnetum-Pinus clades are further reinforced by cpDNA structural evidence. Branch lengths of Cycas-Ginkgo and Gnetum were consistently the shortest and the longest, respectively, in all separate analyses. However, the Gnetum relative rate test revealed this tendency only for the 3rd codon positions and the transversional sites of the first 2 codon positions. A PsitufA located between psbE and petL genes is here first detected in Anthoceros (a hornwort), cycads, and Ginkgo. We demonstrate that the PsitufA is a footprint descended from the chloroplast tufA of green algae. The duplication of ycf2 genes and their shift into IRs should have taken place at least in the common ancestor of seed plants more than 300 MYA, and the tRNAPro-GGG gene was lost from the angiosperm lineage at least 150 MYA. Additionally, from cpDNA structural comparison, we propose an alternative model for the loss of large IR regions in black pine. More cpDNA data from non-Pinaceae conifers are necessary to justify whether the gnetifer or gnepines hypothesis is valid and to generate solid structural evidence for the monophyly of extant gymnosperms.